In the United States heart attacks, almost entirely attributable to coronary atherosclerosis, account for 20-25% of all deaths. Several medical and surgical therapies are available for treatment of atherosclerosis; however, at present no in situ methods exist to provide information in advance as to which lesions will progress despite a particular medical therapy.
Objective clinical assessments of atherosclerotic vessels are at present furnished almost exclusively by angiography, which provides anatomical information regarding plaque size and shape as well the degree of vessel stenosis. The decision of whether an interventional procedure is necessary and the choice of appropriate treatment modality is usually based on this information. However, the histological and biochemical composition of atherosclerotic plaques vary considerably, depending on the stage of the plaque and perhaps also reflecting the presence of multiple etiologies. This variation may influence both the prognosis of a given lesion as well as the success of a given treatment. Such data, if available, might significantly assist in the proper clinical management of atherosclerotic plaques, as well as in the development of a basic understanding of the pathogenesis of atherosclerosis.
At present biochemical and histological data regarding plaque composition can only be obtained either after treatment, by analyzing removed material, or at autopsy. Plaque biopsy is contraindicated due to the attendant risks involved in removing sufficient arterial tissue for laboratory analysis. Recognizing this limitation, a number of researchers have investigated optical spectroscopic methods as a means of assessing plaque deposits. Such xe2x80x9coptical biopsiesxe2x80x9d are non-destructive, as they do not require removal of tissue, and can be performed rapidly with optical fibers and arterial catheters. With these methods, the clinician can obtain, with little additional risk to the patient, information that is necessary to predict which lesions may progress and to select the best treatment for a given lesion.
Among optical methods, most attention has centered on ultraviolet and/or visible fluorescence. Fluorescence spectroscopy has been utilized to diagnose disease in a number of human tissues, including arterial wall. In arterial wall, fluorescence of the tissue has provided for the characterization of normal and atherosclerotic artery. However the information provided is limited by the broad line width of fluorescence emission signals. Furthermore, for the most part, fluorescence based methods provide information about the electronic structure of the constituent molecules of the sample. There is a need for non-destructive real time biopsy methods which provide more complete and accurate biochemical and molecular diagnostic information. This is true for atherosclerosis as well as other diseases which affect the other organs of the body.
The present invention relates to vibrational spectroscopic methods using Fourier transform infrared (FT-IR) attenuated total reflectance (ATR) and near-infrared (IR) FT-Raman spectroscopy. These methods provide extensive molecular level information about the pathogenesis of disease. Both of these vibrational techniques are readily carried out remotely using fiber optic probes. In particular, a preferred embodiment utilizes FT-Raman spectra of human artery for distinguishing normal and atherosclerotic tissue. Near IR FT-Raman spectroscopy can provide information about the tissue state which is unavailable from fluorescence methods. In situ vibrational spectroscopic techniques allow probing of the molecular level changes taking place during disease progression. The information provided is used to guide the choice of the correct treatment modality.
These methods include the steps of irradiating the tissue to be diagnosed with radiation in the infrared range of the electromagnetic spectrum, detecting light emitted by the tissue at the same frequency, or alternatively, within a range of frequencies on one or both sides of the irradiating light, and analyzing the detected light to diagnose its condition. Both the Raman and ATR methods are based on the acquisition of information about molecular vibrations which occur in the range of wavelengths between 3 and 300 microns. Note that with respect to the use of Raman shifted light, excitation wavelengths in the ultraviolet, visible and infrared ranges can all produce diagnostically useful information. Near IR FT-Raman spectroscopy is ideally suited to the study of human tissue.
Raman spectroscopy is an important method in the study of biological samples, in general because of the ability of this method to obtain vibrational spectroscopic information from any sample state (gas, liquid or solid) and the weak interference from the water Raman signal in the xe2x80x9cfingerprintxe2x80x9d spectral region. The FT-spectrometer furnishes high throughput and wavelength accuracy which might be needed to obtain signals from tissue and measure small frequency shifts that are taking place. Finally, standard quartz optical fibers can be used to excite and collect signals remotely.
Near IR FT-Raman spectroscopy provides the capability to probe biological substituents many hundred microns below the tissue surface. In particular, for atherosclerotic tissue, calcified deposits below the tissue surface are easily discerned. Thus, it becomes possible to detect pathologic conditions which would not be apparent using angioscopic methods, as well as to study the detailed molecular basis of the pathology.
In contrast with electronic techniques, the bands in a vibrational spectrum are relatively narrow and easy to resolve. Vibrational bands are readily assigned to individual molecular groups.
The ATR technique offers several features especially suited to sampling of human tissue in vivo. Being a surface technique, the ATR method can non-destructively probe internal human tissue either by direct contact in a hollow organ (e.g. artery), or by insertion of a needle probe. In the mid-IR region, strong water absorption dominates the spectra of highly hydrated samples such as arterial tissue, obscuring the absorption from other tissue components (see FIG. 8). Accurate subtraction of the strong water absorption from FT-IR ATR spectra is relatively easy and very reliable with the high dynamic range, linearity, stability, and wavelength precision of available FT spectrometers. Furthermore, high quality mid-IR spectra of aqueous protein solutions can be collected with fiber optic ATR probes. Such probes are easily adaptable to existing catheters for remote, non-destructive measurements in vivo. The mid-IR ATR technique allows clinicians to gather precise histological and biochemical data from a variety of tissues during standard catheterization procedures with minimal additional risk.
The present methods relate to infrared methods of spectroscopy of various types of tissue and disease including cancerous and pre-cancerous tissue, non-malignant tumors or lesions and atherosclerotic human artery. Examples of measurements on human artery generally illustrate the utility of these spectroscopic techniques for clinical pathology. Results obtained demonstrate that high quality, reproducible FT-IR ATR spectra of human artery can be obtained with relative ease and speed. In addition, molecular level details can be reliably deduced from the spectra, and this information can be used to determine the biochemical composition of various tissues including the concentration of molecular constituents that have been precisely correlated with disease states to provide accurate diagnosis.
Another preferred embodiment of the present invention uses two or more diagnostic procedures either simultaneously or sequentially collected to provide for a more complete diagnosis. These methods can include the use of fluorescence of endogenous tissue, Raman shifted measurements and/or ATR measurements.
Yet another preferred embodiment of the present invention features a single stage spectrograph and charge-coupled device (CCD) detector to collect NIR Raman spectra of the human artery. One particular embodiment employs laser light in the 810 nm range to illuminate the tissue and thereby provide Raman spectra having frequency components in a range suitable for detection by the CCD. Other wavelengths can be employed to optimize the diagnostic information depending upon the particular type of tissue and the type and stage of disease or abnormality. Raman spectra can be collected by the CCD at two slightly different illumination frequencies and are subtracted from one another to remove broadband fluorescence light components and thereby produce a high quality Raman spectrum. The high sensitivity of the CCD detector combined with the spectra subtraction technique allow high quality Raman spectra to be produced in less than 1 second with laser illumination intensity similar to that for the FT-Raman system also described herein.